Breast cancer is commonly divided into four subtypes based on the presence/absence of three distinct receptors: Luminal A, luminal B, HER2, and triple negative/basal-like. Triple negative breast cancer (TNBC) affects approximately 15% of breast cancer patients and has a poorer prognosis than the other subtypes. A major challenge associated with breast cancer in general and TNBC in particular is the heterogeneity of the disease, both between patients and within individual tumors. This has hampered efforts to develop effective therapies. As pointed out by others the ability to describe tumors at the resolution of single cells will enhance our ability to determine the best treatment options and to anticipate disease outcome. The proposed multidisciplinary research program seeks to develop and apply a technology that simultaneously assesses the intracellular signaling activity of multiple enzymes at the single cell level in TNBC. We'll compare and contrast the influence of 2D and 3D cell culture conditions as well as antagonists and agonists on signaling activity. In addition, we'll evaluate the presence of subpopulations exhibiting unique signaling behavior. The ability to define heterogeneous aberrant signaling behavior at the single cell level on a patient-by-patient basis could address what is widely viewed as the single most pressing need in TNBC: the absence of guidelines to manage patients with triple-negative disease.